Higher Order mAb Aggregate Analysis using New

Innovative SEC Technology

1

Ronald E. Majors, Ph.D.LCGC No. America

West Chester, PA USA

Linda Lloyd, Ph.D.Agilent TechnologiesChurch StrettonUnited Kingdom

WCBP 2016Washington, DC

Top 200 pharmaceutical products by U.S. retail sales (20062012)*

*Charts courtesy of the Njardarson Group, Univ. of Arizona, M. Brichacek, N. McGrath, E. Rogers, J.Morton, L.Batory, R. Bauer, J.A.Wurst, and J.T. Njardarson.

URL: http://cbc.arizona.edu/njardarson/group/top-pharmaceuticals-poster

2006 2012

World Preview Outlook to 2020, EvaluatePharma (2014), In 2020, the top 100 prescription drugs based on biologicals will account for 52% of the sales in the U.S.

(those in RED represent a biopharmaceutical product)

BioPharma trends: implications for liquid phase separations

• Require unique new technologies• Workflow driven

Traditional Pharma shifting to biopharmaceuticals

• Move to UHPLC and smaller particles• Superficially porous particle technologyNeed to speed up the analysis

• Require selectivity, resolution and reproducibility• Minimize degradation caused by analysis Accurate characterization

• Non-denaturing eluents – aqueous (Not RP)Analysis of native biomolecules in biocompatible environments

• LC techniques with MS friendly eluents• Low concentration of volatile salts

Connect LC to higher order LC/MS

• Require complete solution with documentation• Defined workflow solutions

Innovative columns/methods needed for biomolecules

• Need products, workflows suitable for biosimilars• Rapid analysis for reduced development time

Some biologics moving off patent

BioPharmaceutical Product Critical Quality Attribute (CQA)

SECCEX

HIC/CEXSEC

SECGlycan column

Glycan column

CEXGlycan column

HIC/RP

HIC/RP

CEX

Peptide mapping/RP

CEX

Reference: Amgen poster

Aggregation

• Proteins including mAbs are relatively unstable

• Do not always adopt native conformation

• Aggregates may form due to improper production, storage or handling

• Aggregates can be:-Tangled clusters of denatured mAbs- Order structures of native mAbs

• Can have important consequences for the safety and efficacy of biopharmaceuticals

- Will reduce the process yield- Will increase the cost of production- May cause patient immunogenicity

Factors that impact aggregation

Manufacturing ProcessesSteps and Products

• Fermentation• Purification• Formulation• Storage• Shipping• Administration

Stress Conditions

• Heat• Freeze-thaw• Cross-linking• Protein concentration• Formulation change –

pH, salt• Chemical modification• Mechanical stress/

surface

Classical techniques for aggregate determination

WHO has set limit for aggregates as <5%

Size Exclusion Chromatography (SEC)

-Larger molecules spend less time in the pores and elute sooner.

-Smaller molecules spend longer in the pores and elute later.

Size in solution is related to retention time

Molecular weight determination

Apparent molecular weight is determined by running samples of known molecular weight and plotting a calibration curve

AdvanceBio SEC 300Å, 7.8 x 300 mm, 2.7µm

Unknown is β-LactoglobulinMW of 18.4 kD

β-Lactoglobulin dimer, apparentMW of 37 kD

Molecular weight determination

However - separation is based on hydrodynamic volume in solution and not molecular weight shape dependency

Light Scattering methods provide absolute molecular weights and increased sensitivity for aggregates

What does that mean for SEC columns?

• SEC columns must deliver accurate, precise quantitation for mAbsand mAb conjugates

• High resolution for more accurate quantitation

• Faster analysis speeds for delivery to deadlines

• Must be stable for long periods of time and high number of injections

• Should cause no change to sample integrity

• Sensitivity for quantitation at low levels - 1 to 5%

• Methods must be easily transferred to other locations, including QA/QC

“Pain points” for current SEC columns

Limited resolution over molecular size range of interest - incomplete resolution makes automated quantitation more difficult - will compromise reproducibility

SEC method impacts sample integrity - non-specific interactions drive mobile phase selection to achieve size separation and will contribute to poor peak shape, recovery and carry over - will compromise accuracy; also, shear forces may be caused by small porosity frits and particles

Long analysis times – large particle sizes and long columns run at low linear velocity - will compromise lifetime if run faster

Limited sensitivity - larger column IDs impact limits of detection and reduces the accuracy of the quantitation for low level aggregation

SEC columns are not very robust and have limited pressure stability

So, what is a good SEC column?

Need high pore volume and optimum pore size No interaction between

particle surface and samples

Mobile phases that do not impact the sample

composition

Introducing AdvanceBio SEC…a new generation of column technology

• A totally new way to make the base silica particle (fully porous) allowing better control of particle diameter, pore diameter and pore volume

• High degree of particle stability allowing for a robust column able to withstand up to 1500 injections, higher flow rates (speed) and no particulate bleed for LS detector

• Novel bonding chemistry minimizes non-specific interactions allowing improved peak shape, excellent recoveries, high degree of inertness and better quantitation (data accuracy), no or low salt in mobile phase and use of pure aqueous mobile phases (no organic).

• A 2.7µm particle to allow good resolution, moderate pressure (can use 400 bar systems) and use of 2µm frits (ruggedness)

Introducing AdvanceBio SEC…a new generation in column technology

• Initial pore size offering: 130 Å and 300Å

• Column dimensions: 4.6mm and 7.8mm i.d’s.150mm and 300mm lengths

• Guard columns: same i.d’s but 50mm length

• Recommended for proteins and peptides with emphasis on monoclonal antibodies analysis and quantification including higher order mAb aggregates and degradation fragments*

• Suitability for different sample types - modified proteins including ADCs and pegylated proteins*

*with same mobile phase

Column performance report & batch test: AdvanceBio SEC 130Å

Typical operating conditions

Parameter Conditions

Mobile phases Aqueous buffers150 mM phosphate buffer, pH 7.0Aqueous organic mixes

pH 2 - 8.5

Operating temp 20 - 30oC (recommended)80oC (maximum)

Operating pressure

<200 bar per column (recommended)400 bar (maximum)

Flow rate 0.1 - 2.0 ml/min for 7.8 mm ID0.1- 0.7 ml/min for 4.6 mm ID

Protein resolving ranges

0.1 - 100 kD for 130Å5 - 1,250 kD for 300Å

Recommended starting conditions

No compromise in operating conditions

Features of AdvanceBio SEC and resulting benefits

Feature Advantages BenefitsNew high pore volume silica Increased resolution Accuracy of quantitation

300Å Pore size Higher mAb aggregates are not excluded

Quantitation of dimer and higher aggregates in single run

130Å Pore size Resolution of mAb fragments, small proteins, peptides

More information about a wider range of samples

Unique–hydrophilic polymeric bonded phase

Narrow symmetrical peaks Gain accuracy and reproducibility of results

2.7µm particle High resolution with lower back pressure

Performance benefits with HPLC and UHPLC systems

300 mm column length High resolution Increased accuracy of quantitation

150 mm column length Shorter analysis times Increased sample throughput

7.8 mm ID Robust separations Reproducibility

4.6 mm ID Increased sensitivity Able to quantify low level aggregation

To increase speed of analysis Sample: IgG I9640 Reduce the column length from 250 to 150 mmIncrease Flow: 0.5mL/min, 1.0mL/min, 1.5mL/min Normalized and aligned

Run time reduced from 12 to 4 minutes

3X sample throughput

Resolution maintained

0.5 mL/min

1.0 mL/min1.5 mL/min

Reproducibility protein standards:four production batches

Batch RS Oval/Myo6273369 2.12

6273380 2.17

6279525 2.12

6277528 2.12

Oval Myo

No batch or column qualification is

required -Replace and go

Protein StandardsBioRad Gel Filtration test mix

Reproducibility mAb aggregate samples:four production batches

Batch RS Dim/Mono6273369 1.92

6273380 1.99

6279525 1.90

6277528 1.96

MonoDim

Multiple batches available for

customer validation

Target application

Sample integrity: recovery

Injection Monomer Peak Area

1 50.5

2 49.6

3 50.6

4 50.0

5 50.2

Five replicate injections of BSA 0.1mg/mL, 1µL equivalent to 0.1 µg of BSA on column

BSA peak area is consistent even with 0.1 µg on-column loads

• No protein recovery issues observed• Sample integrity maintained• Accurate data

Sample integrity: run-to-run

Injection 1

Injection 1500

2

1

2

1

1. Dimer2. Monomer Injection

1Injection

1500

Rsmonomer/dimer

2.19 2.11

Monomer 73.9% 74.3%

Dimer 12.2% 11.5%

Target application: mAb monomer and aggregate(s)

Reproducible quantitation from injection 1 to injection 1500- reduces rework as column ages

Separations of different sample types using same columns and conditions

Retention Time Peak Area

Samples Mean(min)

RSD Mean(mAU/min)

RSD

RituximabInnovator 8.28 0.04 99.33 1.21

RituximabBiosimilar 8.29 0 100 0

CetuximabInnovator 7.86 0 99.60 0.96

HerceptinInnovator 8.034 0 100 0

ADC (T-DM1) 8.106 0.005 98.91 0.33

Biotherapuetic mAbs, innovators, biosimilars and ADC

The same column [AdvanceBio SEC-3, 300Å, 7.8 x 300 mm, 2.7 μm] and mobile phase: [(PBS) 50 mM sodium phosphate, 150 mM sodium chloride, pH 7.4]for mAbs and ADCs - Eliminates stocking multiple column types - Eliminates multiple methods

min2 4 6 8 10 12 14

mAU

0

200

400

600

800

1000

1200

1400

1600

220nm, Heat/pH stressed ADC220nm, Intact ADC

Aggregated ADC

Intact ADC

Degraded ADC

min2 4 6 8 10 12 14

mAU

02004006008001000120014001600

RituximabInnovator

min2 4 6 8 10 12 14

mAU

02004006008001000120014001600 Rituximab

Biosimilar

min2 4 6 8 10 12 14

mAU

0100200300400500600700 , Cetuximab

Innovator

min2 4 6 8 10 12 14

mAU

02505007501000125015001750 Herceptin

Innovator

A B

C D

Different types of samples: complex conjugated proteins

PEGylated Proteins - increase bioavailability, increase serum half-life, decrease immunogenicity

Pegfilgrastin (PEG GCSF)

Summary: AdvanceBio SEC delivers

Accuracy of quantitation:• Unique chemistry: improved peak shapes with high efficiency for increased resolution and

provides better sensitivity for low level aggregate/fragment analysis.• Unique chemistry: reduced non-specific interactions over a wide range of pH and salt

concentrations to maintain sample integrity and provide high data accuracy• Narrow particle size distribution and high pore volume: increased column efficiency and

resolution• Increase resolution: run two columns of the same pore size in series increases resolution • Extended resolving range: run two columns of different pore size in series and

enable analysis of multiple attributes in one run• Decreased analysis time: run columns at higher flow rates; increases sample throughput

Method robustness:• Particle integrity: reduces soluble and insoluble column bleed for mass spec and light

scattering detectors • Control of manufacturing process: ensures batch-to-batch and column-to-column

reproducibility• Particle size and 2µm frit: reduces the risk of column clogging and early failure; operates

at lower pressure.• Increased pressure stability: reduces risk of column collapse with high viscosity eluents

or during solvent transfers• QA testing: application specific protein test mix

Acknowledgements

o Thanks to co-author Linda Lloyd for helping to put together the presentation.

o Thanks to Agilent R/D and Applications’ chemists in Church Stretton, UK and Wilmington, DE, USA for developing an innovative product and for generating the technical data.